Isolation and Identification of Effective Probiotics on Drug-Resistant Acinetobacter baumannii Strains and Their Biofilms

Introduction This study aimed to identify, assess, and isolate strong lactobacilli demonstrating broad antibacterial and anti-biofilm activity against drug-resistant strains of Acinetobacter baumannii. Additionally, the mechanism of inhibition of these organisms was to be determined. Methods Over a 6-month period (from December 2021 to June 2022), 53 clinical A. baumannii strains were collected from clinical samples. Twenty probiotic strains were isolated from local dairy products. Antibacterial activity of Lactobacillus strains' cell-free supernatant (CFS) was identified using the agar well diffusion method and the microbroth dilution test. Anti-biofilm effect was performed by the microtiter plate assay. The MTT assay was also used to look into the probiotics' cytotoxic effects on the L929 fibroblast cell line. Results During the 6-month period, 53 clinical A. baumannii strains were obtained and identified. Out of 20 lactobacillus strains, the CFS of a lactobacillus strain (named L9) showed an inhibitory effect against all A. baumannii strains. Using the broth microdilution method, it was shown that the minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) of CFS extracts of L9 strains against A. baumannii strains were both ¼ mg/mL. The result of the anti-biofilm showed that the selected probiotic could inhibit biofilm formation. The most common organic acid produced by all Lactobacillus strains, according to the HPLC method, was lactic acid, which was followed by acetic acid. The L929 fibroblast cell line was used in the cytotoxicity assay, which revealed that 100% of the cells in the L929 fibroblast cell line survived treatment with successive doses of CFSs for a full day. Conclusion The probiotic strain isolated from local yogurt in this study showed potential anti-biofilm and antimicrobial properties against all drug-resistant Acinetobacter isolates. Given the increasing interest in probiotic microorganisms based on their high health benefits, further studies are recommended on the mechanisms of action between probiotics and A. baumannii strains to find new solutions for biological control and treatment of these infections without the use of antibiotics.


Introduction
Worldwide, the prevalence of multidrug-resistant (MDR) microorganisms is rising, presenting a major health concern to individuals [1,2].Hospital-acquired infections caused by MDR strains have resulted in increased treatment costs, mortality, and morbidity, thereby compromising patient safety.In response to the surge in MDR bacteria and the constraints on antibiotic use due to their side efects, researchers are exploring potential alternatives [3,4].Living microorganisms, such as probiotic bacteria like lactobacilli, play a pivotal role in human health and constitute the most signifcant component of both human and animal gut microfora [5].Te probiotic microorganisms can inhibit pathogenic organisms' growth and pathogenicity.Lactobacillus species are known to generate a diverse range of antimicrobial agents, such as bacteriocins, lactic acids, acetic acids, and other substances, which are found in the culture supernatant of these microorganisms [6].
Acinetobacter baumannii is the most common and harmful nosocomial pathogen causing human infections globally, particularly in critical care units (ICUs) [7].Infections induced by A. baumannii commonly lead to substantial rates of morbidity and mortality, reaching up to 60%, including ventilator-associated pneumonia (VAP), septicemia, meningitis, endocarditis, urinary tract infection (UTI), keratitis, and ophthalmitis [8]. A. baumannii has rapidly progressed from MDR to extremely drug-resistant (XDR) due to its evolving antibiotic resistance [9].
Although prescription drugs like carbapenems and fuoroquinolones still exhibit efectiveness against XDR A. baumannii, the minimum inhibitory concentrations (MICs) have increasingly risen, and nearly carbapenemsresistant A. baumannii have been documented [10].Despite this trend, carbapenems remain the preferred antibiotic therapy for Acinetobacter infections.Te lack of efective antibiotics for A. baumannii infection highlights the need for alternative therapies [11].
Te extracellular polysaccharide matrices (EPS) that bacteria self-synthesize serve as a protective measure against bioflms [12].Te presence of bioflms introduces various challenges in the medical feld, impeding the clinical treatment of persistent infections associated with various indwelling medical devices, as well as diseases connected to chronic and wound-associated illnesses.Research has indicated that the administration of probiotics can assist in the prevention and/or treatment of infections linked to bioflms [13,14].
It has been discovered that administering probiotics can help prevent and/or treat infections linked to bioflms.Te creation of bioflm and bacterial (pathogenic) MDR causes antibiotics to be inefective in treating infection [15].Probiotic lactobacilli, including Lactobacillus rhamnosus, seem to have strain-specifc antibacterial action, though [16].Due to its signifcance, the objectives of this work were to identify, evaluate, and isolate potent lactobacilli with wide antibacterial and anti-bioflm activity against drug-resistant A. baumannii strains, as well as to identify the mechanism of inhibition of these organisms.

Sample Collection and Identifcation.
A. baumannii were collected from clinical samples, over a six-month period (December 2021 to June 2022).Te specimens included respiratory, blood, wounds, burns, surgery, urine, and cerebrospinal fuid (CSF) samples obtained from hospitalized patients in three hospitals in the province of Isfahan (Al-Zahra, Amin, and Milad).Tese samples were cultured on MacConkey agar and blood agar medium (HiMedia, India) and then were incubated for 24 hours at 37 °C.Gram staining and biochemical testing were used to identify the pure strains [17].

Isolation and Identifcation of Lactobacillus Strains.
Samples were collected from various local sources to obtain 20 probiotic strains from local dairy products, including sheep yogurt, cow yogurt, camel milk, cow's milk, sheep's milk, goat's milk, and native buttermilk from diferent regions of Isfahan province in Iran, namely, Shahreza, Golpayegan, Semirom, Fereydunshahr, and Najafabad.For bacterial isolation, 1 mL of each dairy sample underwent homogenization, was subsequently suspended in a 2% w/v sodium citrate solution (Merck, Germany), and was then introduced into 10 mL of MRS broth (HiMedia, India).After 48 hours, 0.02 mL of the solution was spread over MRS agar media following a 24-hour incubation at 37 °C [18].

Evaluation of Lactobacillus Strains' Antibacterial Activity.
Agar well difusion and broth microdilution tests were performed to detect antibacterial activity of Lactobacillus strains.

Agar Well Difusion Method.
As previously mentioned, the cell-free supernatant (CFS) of Lactobacillus cultures was extracted and utilized in the agar well difusion procedure [20].To assess antibacterial activity, the growth inhibition zones surrounding the wells were measured following a 24hour incubation at 37 °C.Tese inhibition zones were then compared with those observed in the control group.

Broth Microdilution Assay.
To determine the antibacterial activity (MIC and MBC) of CFS of probiotics against clinical isolates of MDR A. baumannii, a broth microdilution test was employed in accordance with previous descriptions [20].In brief, 100 μL of the diluted (½, ¼, 1/8, and 1/16) CFS of Lactobacillus was transferred to 96-well plates in the presence of MRS broth medium.A 96-well plate containing the produced suspension (10 8 CFU/ml) was then cultivated and incubated for 24 h at 37 °C.After that, it was cultured on blood agar medium and kept at 37 °C for another 24 hours [21].Te positive and negative controls were the wells devoid of bacteria and extract, respectively.By measuring optical density (OD470 nm), MIC and MBC, representing the lowest concentrations of CSF capable of inhibiting the growth of the pathogen and eradicating all pathogenic bacteria, were determined [22].

2
Canadian Journal of Infectious Diseases and Medical Microbiology 2.7.Time-Kill Test in Cocultures.By coculturing in microtiter plates, the probiotic was tested for its ability to kill the Acinetobacter strain after a minimum period of time.Lactobacillus and A. baumannii isolates were cultivated in trypticase soy broth (TSB) and MRS broth, respectively.Once the A. baumannii strain had been diluted to a 0.5 McFarland turbidity, a suspension of the isolates was prepared.Subsequently, a mixture comprising 100 μL of cell-free supernatant (CFS) from a coculture of Lactobacillus strains and 100 μL of A. baumannii was prepared in a 96-well microtiter plate and incubated for 24 hours at 37 °C.To assess the inhibitory or bactericidal efects, aliquots from the coculture suspension were cultured on blood agar at 1-hour intervals (1 h, 2 h, 4 h, 8 h, 12 h, 24 h, and 48 h) and incubated at 37 °C for 24 hours [23].Each experiment was conducted in duplicate and repeated three times.

Assessment of Acid Tolerance of Probiotic.
Te Lactobacillus strains were inoculated into MRS broth and incubated for 48 h at 37 °C.Ten, the Lactobacillus strains were inoculated into PBS (pH1, pH2, and pH3, pH4 (control)) and PBS (pH4 as control) and incubated for 0, 30 min, and 1 hour, at 37 °C.After that, MRS agar was spread out to a density of 50 g/ml, and it was incubated at 37 °C for 24 h.Te vitality of Lactobacillus strains exposed to normal conditions and acidic conditions (pH1, pH2, and pH3) was used to measure the acidic tolerance.Tis experiment was repeated in duplicate.
2.9.Anti-Bioflm Efect of Lactobacilli.For this purpose, the microtiter plate test was utilized.Initially, the bacteria were cultured in MRS medium for one day at 37 °C.Suspensions (0.5 McFarland standard) from this culture were then inoculated into MRS medium (supplemented with 0.2% sucrose) containing ½, ¼, and 1/8 MICs of the CFS extracts.A volume of 200 μL of this solution was added to each well of a 96-well microplate.Te positive and negative controls were represented by wells without microorganisms and without CFS, respectively.Te microplate was incubated at 37 °C for 24 hours.Subsequently, the wells were emptied, and crystal violet was added, followed by cleaning with 95% ethanol.Finally, the optical density (OD) of crystal violet associated with bioflms was measured at a wavelength of 570 nm.Te experiments were performed three times, utilizing A. baumannii ATCC 196006 as a positive control and an uninfected medium as a negative control [22].

Determining the Possible Inhibitory Mechanism.
Te test was performed to determine whether the inhibitory and antimicrobial efects of the CFS of probiotics were due to the presence of organic acids or other mechanisms.Terefore, to probiotics with pH 4, add 4 drops of NaOH (sodium hydroxide) to neutralize (pH 7), and then in a tube of neutral probiotics and in the other tube, the main probiotic supernatant is placed.Agar well difusion method on the Muller Hinton agar used as previously described.Te plates were cultivated with the 0.5 McFarland turbidity (A.baumannii), create four wells with a sterile glass Pasteur pipette and fll two of them with neutral probiotics (pH�7) and two of them with main probiotics (pH�4), then plates were incubated at 37 °C for 24 h.In parallel, the suspension equivalent to 0.5 McFarland turbidity of Acinetobacter (in a volume of 1000 microliter) was poured into a sterile tube with a screw cap, and the same volume of neutral probiotics was added.Te tube was then placed in the incubator.After 4 h, 50 microliter of it was cultured on chocolate agar medium at 37 °C for 24 h [18].

High-Performance Liquid Chromatography (HPLC).
Te identifed Lactobacillus strains were cultured in MRS broth medium for 72 hours, after which the culture was centrifuged at 10,000 g for 10 minutes.Following centrifugation, the supernatant was separated from the bacterial pellet and then fltered through a 0.25 µm syringe flter.
To confrm the sterility of the fltrate and absence of Lactobacilli growth, a reculturing step in MRS broth medium was conducted for an additional 72 hours.HPLC was carried out using twenty microliters of CFS, employing a fow rate of 1 mL•min −1 , maintaining pH at 3.6, and utilizing reversed-phase HPLC columns (C18, 25 cm × 4.6 mm) with an aqueous mobile phase (phosphate bufer-CH3CN 10 mM). Figure 1 shows the (UV) absorbance measured at room temperature at 25 °C [24].
2.12.MTT Assay.Purchased from the Pasteur Institute in Tehran, Iran, normal subcutaneous connective tissue (L929) cell lines were cultivated in DMEM low glucose medium with 10% FBS and antibiotics (including 50 μg/ml of streptomycin and 50 U/ml of penicillin).Te cells were incubated at 37 °C with 5% CO2 and 90% humidity.After 24 h, 20 microliter of the probiotic supernatant was fltered twice with a 0.22-micron syringe flter.Ten, added and dilution was done in 5 dilutions 1, 0.5, 0.25, 0.125, and 0.062.Te microplates were then incubated at 37 °C for 24hours.Each research group's cells were incubated at 37 °C for 4 h after being exposed to 50 μL of the MTT reagent (5 mg/ml in sterile PBS).To dissolve the formazan crystals, 50 μl of DMSO was added after the culture media had been taken out.Te fndings were determined using a microplate reader from Bio-Tek Instruments, Inc. of Vermont, USA, at an absorbance of 570 nm [25,26].
Te percentage of cell viability was calculated using where A 570 : the absorbance at 570 nm; sample: a monolayer of every cell line plus diferent treatment concentrations in RPMI media; blank: diferent treatment concentrations in RPMI medium; and control: a monolayer of every cell line plus RPMI medium with no modifcations.
2.13.Identifcation of Selected Lactobacilli.DNA was extracted from pure cultures made from bacterial colonies and stored at −20 °C as previously described.By using the universal primers (CinnaGen Co, Iran) [18], the bacteria identifed using these traditional tests were verifed.
Using the following steps, the 16S rRNA gene was amplifed: initial denaturation at 95 °C for 5 minutes, 30 cycles of denaturation at 95 °C for 30 seconds, annealing at 54 °C for 30 seconds, extension at 72 °C for 30 seconds, and a fnal extension cycle at 72 °C for fve minutes.Direct sequencing was employed at Bio Magic Gene, Inc., Karaj, Iran, to locate the nucleotide sequences of successful PCR products.Te sequences were matched with the NCBI's BLAST search results and then registered.

Probiotics Isolation and Identifcation.
Based on our fndings, 20 Lactobacillus strains were identifed local yogurt and milk samples, and their antimicrobial properties against strains of A. baumannii were taken into account.Out of 20 Lactobacillus strains, the CFS of one strain (L9) demonstrated an inhibitory efect (showing inhibition zones with a diameter of 14 mm) against every strain of A. baumannii in the well difusion method, according to the results.
For this reason, in the remaining trials, the CFSs of the L9 strain were employed.
After 24 hours of incubation, the results showed that L9 was able to grow at pH 1, pH 2, and pH 3 after zero, 30 minutes, and one hour despite variations in the degree of viability.In this test, pH 4 is considered as a positive test.

Antimicrobial Efect of Probiotics against A. baumannii.
Using the broth microdilution method, it was shown that the MICs and MBCs of CFS extracts of L9 strains against A. baumannii strains were both ¼ mg/mL and the ratio of the concentration of MIC and MBC was similar.
Furthermore, no change in OD was observed in the fndings of the liquid coculture assay, and after 24 h, the isolates of A. baumannii demonstrated 100% suppression of growth.Tis demonstrated that all A. baumannii growth was suppressed by L9 Lactobacillus strains.Additionally, when A. baumannii strains were inoculated and cultured in blood agar medium, no growth of those strains was seen as compared to positive controls of A. baumannii strains without lactobacilli coculture (the killing efect was 100%).Moreover, the minimum time required to inhibit A. baumannii strains is only one hour by probiotics, which shows the potential properties of probiotics.Also, L9 strains exhibited tolerance to acid (pH 1, 2, and 3) after 0, 30 min, and 1 hour, respectively (Figure 3).
3.4.Antibiotic Susceptibility.Among the antibiotics tested, the L9 strain was sensitive to ampicillin, penicillin, tetracycline, linezolid, rifampin, erythromycin, ciprofoxacin, clindamycin, nitrofurantoin, and gentamicin and resistant to teicoplanin, trimethoprim, and vancomycin.Canadian Journal of Infectious Diseases and Medical Microbiology 3.5.Anti-Bioflm Activity of Probiotic.Te outcome of the probiotics' anti-bioflm inhibitory efect demonstrated that, at concentrations of 1, ½, and ¼, the chosen probiotic was able to inhibit bioflm formation and prevent the formation of A. baumannii strains; however, at concentrations of 1/8 and 1/16, A. baumannii strains formed bioflm, and the probiotic strain was unable to inhibit them (Figure 4).Investigation of probable inhibition mechanism of L9 strains showed that in contrast to pH 7, in pH 4, the CSF of strain L9 had inhibition zones against A. baumannii isolates in the well difusion method.On the other hand, neutralized supernatants (pH 7) of L9 strains did not have any inhibitory activity against A. baumannii, which showed that the inhibitory efects of the L9 strains were due to their organic acid production.
Trough the use of HPLC, an assessment of the variety and amount of organic acids generated by diferent Lactobacillus strains revealed that lactic acid and acetic acid were the dominant organic acids produced by all strains.Te concentrations of lactic acid and acetic acid were 3.2 gr/ml and 0.5 gr/ml, respectively (Figure 1).
Te cytotoxicity test against the L929 fbroblast cell line showed that cell viability of L929 fbroblast cell following treatment with serial concentrations of CFSs for 24 h was 100%.Tese results were not diferent from the control bacteria sample (100%).

Discussion
Globally, MDR-A.baumannii strains causing nosocomial infections lead to a growing concern due to high mortality rates and limited treatment options [27,28].Most of their important features include survivability in the hospital environment and rapid resistance to various antibiotics.Terefore, alternative treatment methods, such as probiotics, are sought to prevent or treat nosocomial infections [29].
In the present study, A. baumannii strains were isolated from various clinical samples to evaluate their antibiotic susceptibility patterns.Additionally, diferent probiotic strains were isolated from local dairy products to identify a strain with efective antimicrobial activity against Acinetobacter strains and investigate their probiotic benefts.
In this study, a large number of A. baumannii strains were isolated from various clinical samples, including blood, respiratory secretions, wounds, and urine, respectively, which almost corresponds to studies conducted until now [30,31].Te results of this study, along with other investigations on the isolation of A. baumannii strains, suggest a higher prevalence of this bacterium in respiratory secretions, blood, wounds, and urine samples.
In a study conducted in Mashhad [16], all isolates were MDR, with imipenem, meropenem, ceftazidime, ceftriaxone, cefxime, cefotaxime, and cephalexin showing the greatest level of resistance (100%) among the isolates.Conversely, the least resistance (19.4%) was found against polymyxin B. Tis study's outcomes support our conclusions [16].Similarly, in line with the present study, Nouri et al. and Azimi et al. presented evidence of low sensitivity to meropenem and imipenem in A. baumannii isolates, which were resistant to these antibiotics to a large extent [36,37].
An Iranian meta-analysis examined antibiotic resistance patterns among isolates of A. baumannii from ICUhospitalized patients.Te results revealed that the most antibiotic resistance was observed to ceftazidime, ceftriaxone, cefxime, cefotaxime, imipenem, and meropenem, while colistin and polymyxin B exhibited the highest sensitivity.Te results of the present study are in agreement with this review and meta-analysis [38].
Contrary to our results regarding the high resistance of the isolates to piperacillin-tazobactam, Azimi et al. reported high sensitivity to this antibiotic [37,38].Te discrepant results of this study with the mentioned studies can be attributed to the type of examined samples, the use of diferent antibiotic disks, and variations in geographical locations.Additionally, the overuse of this antibiotic in hospitals could explain the discrepancy.
Lactic acid-producing bacteria are probiotics with antiinfammatory and antibacterial properties that have been mostly studied in various felds.Lactobacilli, belonging to the Lactobacillaceae family, are the most important and frequently studied microorganisms within this family, encompassing numerous identifed species [39].
Te results of the agar well difusion method showed that only one probiotic isolated from local yogurt could create growth inhibition zones on all A. baumannii strains.Molecular studies confrmed the identifcation of our selected probiotic as L. rhamnosus, determined through biochemical and molecular PCR tests.In this research, L. rhamnosus exhibited a remarkably broad-spectrum activity with inhibitory and lethal efects on all strains of the A. baumannii pathogen.Te examined probiotic demonstrated a highly efective inhibitory impact in the microtiter plate and coculture method and in determining MIC and MBC.Notably, the MIC and MBC values were equal to ¼ concentration after the coculture of this probiotic bacterium with Acinetobacter strains.Tis means that the probiotic could inhibit and, additionally, kill the pathogen at this concentration, indicating the potential value of L. rhamnosus.Te results of the lethal time test also showed that the studied probiotic could kill the Acinetobacter strains only after one hour, which is a valuable result.
Probiotic bacteria have benefcial antibacterial efects through bioflm formation [40].Tis study evidenced the strong anti-bioflm efects of L. rhamnosus, efectively preventing replacement, adhesion, and bioflm development by Acinetobacter strains through its own bioflm formation.Te inhibitory efect of probiotics on bioflm formation by pathogenic strains has been investigated in numerous research studies.
In a research conducted in India, it was documented that Lactobacillus gasseri, obtained from the feces of infants, exhibited antagonistic and antimicrobial properties against pathogenic bacteria such as Staphylococcus aureus, Enterococcus faecium, Enterobacter, Klebsiella pneumoniae, A. baumannii, and Pseudomonas aeruginosa [41].
Sultan Dalal et al. reported the antagonistic activity of L. plantarum and L. fermentum isolated from the feces of healthy infants, against nosocomial infections caused by A. baumannii and P. aeruginosa [42].Te antimicrobial efect of L. plantarum and L. piscium strains isolated from goat milk against A. baumannii was reported by Fezoni et al. [43].According to the promising antimicrobial efects of these probiotics, goat milk could be used as an adjuvant treatment for these infections [43].
In a study conducted in the United States [44], topical treatment with the supernatants of Lactobacillus acidophilus, L. casei, and L. reuteri was used on mouse wounds infected with A. baumannii.Te results showed that the antimicrobial and anti-infammatory efects of probiotics with local treatment could enhance wound healing [44].Te study, in general, demonstrated that the topical application of some Lactobacillus species can be efective against the Gramnegative pathogen A. baumannii.
Guan et al. conducted a study on the antibacterial properties, specifcally against Bacillus subtilis and Salmonella enterica and an anti-bioflm of L. rhamnosus.Te antibacterial activity of L. rhamnosus cells was diferent 6 Canadian Journal of Infectious Diseases and Medical Microbiology under diferent culture conditions, and the intensity of antibacterial efects was found to be unrelated to the biomass.Additionally, an isolated cell-surface extract revealed a wide spectrum of antimicrobial and anti-bioflm capacity.
Te main components of the extract were identifed as polysaccharides and proteins.Te properties of the extract indicated that it might be a type of biosurfactant [44].
In another study, CFS extracts of L. casei and L. rhamnosus were found to be exhibiting antagonistic and anti-bioflm efects against S. aureus.Tis fnding suggests that future research could lead to the development of drugs derived from these CFSs to combat S. aureus infections [45].
Te use of probiotics and their metabolites presents a promising strategy to prevent bioflm growth by various pathogenic microorganisms.In the present study, in addition to the appropriate antimicrobial and anti-bioflm properties of the L. rhamnosus strain, other investigated properties include the bile-esculin test, 6.5% salt tolerance, catalase, DNase, hemolysis, CAMP, and cold enrichment, as well as the acid resistance assay and MTT test.
A negative 6.5% salt tolerance test was reported in the current study, consistent with de Vries et al. who reported a low growth of Lactobacilli in a salt concentration of ≥5%, and the survival percentage decreased at high salt concentrations [46].
Te inhibitory mechanism of the L. rhamnosus strain was evaluated in this investigation.To confrm the presence of organic acids in L. rhamnosus, the potential inhibitory mechanism was initially determined.
Te results showed that the probiotic's inhibitory activity of the probiotic was due to the presence of strong organic acids.In another step, the presence of acetic acid and lactic acid and their concentrations were evaluated using HPLC.Te analysis of organic acids in the L. rhamnosus supernatant revealed levels of 0.5 g/l for acetic acid and 3.2 g/l for lactic acid.Tese results strongly suggested that the inhibitory activity of L. rhamnosus was primarily due to the presence of organic acids, namely, acetic acid and lactic acid.

Conclusion
In this study, the probiotic strain isolated from local yogurt showed potential anti-bioflm and antimicrobial properties against all drug-resistant Acinetobacter isolates.Given the increasing interest in probiotic microorganisms due to their signifcant health benefts, further studies are recommended on the mechanisms of action between probiotics and A. baumannii strains to fnd new solutions for biological control and treatment of these infections without the use of antibiotics.